The cathepsin family of lysosomal protease includes the cysteine protease; cathepsins B, H, K, L, O2, and S, and the aspartyl protease; cathepsins D, and G. The various members of this endosomal protease family are differentially expressed. Some, such as cathepsin D, have a ubiquitous tissue distribution while others, such as cathepsin L, are found only in monocytes, macrophages, and other cells of the immune system.
The cathepsins represent the major endopeptidases in the lysosome (Huisman W, et al. (1974) Biochem Biophys Acta 370:297-307) and as such participate in the degradation of proteins entering the vacuolar system by endocytosis and in the turnover of cytoplasmic proteins. The cathepsins are also active in 1) initiation of proteolytic cascades by proenzyme activation, 2) processing of the II alpha beta heterodimer in endosomes for expression of functional MHC class II molecules which bind antigenic peptides, and 3) processing of antigen in antigen-presenting cells. The secreted forms of several members of this family function in tissue remodeling through degradation of collegen, laminin, elastin, and other structural proteins of basement membranes (Mizuochi T, (1994) Immunol Lett 43:189-193, Baldwin E T, (1993) Proc Natl Acad Sci 90:6796-6800). Both cysteine and aspartyl cathepsins are used by various parasitic protozoa for the catabolism of host cell proteins and/or facilitating host invasion (Becker M M et al. (1995) J Biol Chem 270: 24496-24501, Rosenthal P J et al. (1988) J Clin Invest 82:1560-1566, McKerrow J H (1993) Annu Rev Micrbiol 47:821-853).
The various cathepsin proteases differ in their gene structures and in their transcriptional regulation. The cathepsin D gene promoter has a compound structure with features of both housekeeping genes (high G+C content and potential transcription factor SP-1 sites) and regulated genes (TATA sequence). RNase protection assays show that transcription is initiated at five major transcription sites (transcription site-I to transcription site-V) spanning 52 base pairs. Site-directed mutagenesis studies indicate that the TATA box is essential for initiation of cathepsin D gene transcription at transcription site-I. This suggests that cathepsin D behaves, depending on the conditions, as a housekeeping gene with multiple start sites or as a hormone-regulated gene that can be controlled from its TATA box (Cavailles V (1993) Proc Natl Acad Sci 90:203-207). The cathepsin L gene promoter has no TATA box but includes several SP-1 sites, two AP-2 transcription regulatory element binding sites (a promoter under the control of the tumor promoter and cAMP), and a cAMP response element. Experimental data confirm that the expression of cathepsin L is induced by malignant transformation, growth factors, tumor promoters, and cyclic AMP (Troen B et al.(1991) Cell Growth Differ 2:23-31).
Altered regulation and expression of these two different cathepsins is evident in disease states. Cathepsin D is overproduced and hypersecreted by breast cancer cells. Clinical studies show a strong correlation between high concentrations of cathepsin D in the cytosol of primary tumor cells and further occurrence of metastasis. Cathepsin D is expressed at high levels in hormone independent breast cancer, is induced by estrogen in hormone dependent breast cancer, and appears to be independent of other more classical prognostic factors. In nude mice, transfection of cathepsin D cDNA into tumor cells increases their metastatic potential, suggesting that overexpression of this protease may be one of the factors responsible for metastasis (Rochefort H (1992) Acta Oncol 31:125-30, Long B J (1996) Cancer Lett 99:233-238). The mechanism by which this protease might facilitate metastasis is not fully characterized, although cathepsin D has the potential to initiate a proteolytic cascade, to degrade extracellular matrix and to liberate growth factors from the matrix. In vitro studies have noted that transfected cathepsin D stimulates high density cancer cell growth via an intracellular mechanism leading to a decreased secretion of growth inhibitors (Liaudet E (1995) Cell Growth Differ 6:1045-1052). Altered cathepsin D levels are present in biopsy specimens in prostate and bladder carcinomas and are shown to correlate with tumor grade (Ross J S (1995) Am J Clin Pathol 104:36-41, Dickinson A J (1995) J Urol 154:237-241).
Altered cathepsin activity and/or distribution may play a role in certain brain diseases. In Alzheimer's disease, A4 amyloid peptide, the main constituent of amyloid plaques and cerebrovascular amyloid deposits, derives from a large amyloid precursor protein (APP) by the action of endoproteases which release the amino and carboxyl termini to generate the aggregating form of A4. In the brains of Alzheimer's patients, more than 90% of the pyramidal neurons in lamina V and 70% in lamina III displayed 2- to 5-fold elevated levels of cathepsin D mRNA by in situ hybridization compared with neurologically normal controls. An endogenous protease activity from diseased samples was found to be active in acidic conditions and inhibited by pepstatin, two characteristics of cathepsin D, suggesting that a cathepsin D-like activity from human brain may be responsible for APP processing (Evin G (1995) Biochemistry 34:14185-14192, Cataldo A M (1995) Neuron 14:671-680). There is a significant increase in cathepsin D activity in biopsies from Huntington's disease (Mantle D (1995) J Neurol Sci 131:65-70) and there are increased levels of cathepsin D mRNA in scrapie-infected mice (Diedrich J F (1991) J Virol 65:4759-4768).
Abnormal regulation of cathepsins is observed in several inflammatory disease states. In fibroblastoid synovial lining cells isolated from rheumatoid and other chronic inflammatory synovial tissues, the mRNA for stromelysin, vimentin, IL-4, IL-6, TIMP-1, cathepsin D, gelatinase, TGF alpha, c-fms and DR beta is preferentially expressed. This modulated pattern of gene expression is correlated with the phenotype of this inflammatory condition (Ritchlin C et al. (1994) Scand J Immunol 40:292-298). Cathepsin L and D expression was evaluated in synovial tissues from patients with rheumatoid arthritis (RA) and osteoarthritis (OA), using in situ hybridization with digoxigenin-labeled RNA probes. Both RA and OA synovial tissue expressed cathepsins L and D. The expression of the cathepsins was markedly higher in interstitial regions and in perivascular infiltrates of RA synovial tissue compared with OA specimens. The differential expression of cathepsins L and D mRNA in RA and OA synovial tissues supports the concept that these enzymes may contribute to the influx of mononuclear cells into the synovium and suggests that the adhesion of synovial cells to cartilage mediates the invasive/destructive process in RA (Keyszer G M (1995) Arthritis Rheum 38:976-984.
The cathepsins are believed to be involved in several other diseases states. In an experimental model of human glomerular disease, the administration of a specific, irreversible inhibitor of cysteine protease (trans-epoxysuccinyl-L-leucylamido-3-methyl-butane) significantly reduces proteinuria in rats (Baricos W H (1991) Arch Biochem Biophys 288:468-72.) The fibroblasts from patients with mucolipidosis II and III have a severely compromised capacity for endogenous lysosomal protein degradation that appears to result from multiple cathepsin deficiencies (Kopitz J (1993) Biochem J 295 (Pt 2): 577-580). The platelet aggregating cysteine protease implicated in thrombotic thrombocytopenic purpura shows the characteristics of a lysosomal cathepsin (Consonni R (1994) Br J Haematol 87:321-324).
Cathepsin D knockout mice develop normally during the first 2 weeks, stop thriving in the third week and die in a state of anorexia at day 26.+-.1. An atrophy of the ileal mucosa observed in the third week progresses towards widespread intestinal necroses accompanied by thromboemboli. The thymus and spleen undergo massive destruction with loss of T and B cells. The lysosomal bulk proteolysis is, however, maintained. These results suggest that the major functions of cathepsin D involve limited proteolysis of proteins regulating cell growth and/or tissue homeostasis (Saftig P, (1995) EMBO J 14:3599-3608).
Cathepsins have a role in processes that involve proteolysis of specific proteins and tissues in local microenvironments including inflammation, metastasis and peptide and proenzyme processing. The increased expression and differential regulation of these protease is linked to the metastatic potential of a variety of cancers and as such is of therapeutic and prognostic interest. Evidence of the involvement of cathepsins associated with protein processing in diseases such as Alzheimer's disease, Huntington's disease, mucolipidosis and arthritic inflammation suggests that modulation of the cathepsins may ameliorate these disease processes. The polynucleotide sequences and proteins of the claimed invention would satisfy this need by providing the means for diagnosis, study, prevention and treatment of these diseases.